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Quantitative assessment of short amplicons in FFPE-derived long-chain RNA

View Article: PubMed Central - PubMed

ABSTRACT

Formalin-fixed paraffin-embedded (FFPE) tissues are important resources for molecular medical research. However, long-chain RNA analysis is restricted in FFPE tissues due to high levels of degradation. To explore the possibility of long RNA quantification in FFPE tissues, we selected 14 target RNAs (8 mRNAs and 6 long noncoding RNAs) from literatures, and designed short (~60 bp) and long (~200 bp) amplicons for each of them. Colorectal carcinomas with adjacent normal tissues were subjected to quantitative reverse-transcription PCR (quantitative RT-PCR) in 3 cohorts, including 18 snap-frozen and 83 FFPE tissues. We found that short amplicons were amplified more efficiently than long amplicons both in snap-frozen (P = 0.0006) and FFPE (P = 0.0152) tissues. Nonetheless, comparison of colorectal carcinomas with their adjacent normal tissues demonstrated that the consistency of fold-change trends in a single short amplicon between snap-frozen and FFPE tissues was only 36%. Therefore, we innovatively performed quantitative RT-PCR with 3 non-overlapping short amplicons for 14 target RNAs in FFPE tissues. All target RNAs showed a concordance of 100% of fold-change trends in at least two short amplicons, which offers sufficient information for accurate quantification of target RNAs. Our findings demonstrated the possibility of long-chain RNA analysis with 3 non-overlapping short amplicons in standardized-preserved FFPE tissues.

No MeSH data available.


Comparisons of Ct values obtained from short and long amplicons in frozen and FFPE tissues.(a): Comparison of Ct values obtained from short and long amplicons in frozen tissues. (b): Comparison of Ct values obtained from short amplicons in frozen and FFPE tissues. Frozen-short A: quantification with short-A amplicons in frozen tissues. FFPE-short A: quantification with short-A amplicons in FFPE tissues. Error bars indicate the SD of the Ct values.
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f2: Comparisons of Ct values obtained from short and long amplicons in frozen and FFPE tissues.(a): Comparison of Ct values obtained from short and long amplicons in frozen tissues. (b): Comparison of Ct values obtained from short amplicons in frozen and FFPE tissues. Frozen-short A: quantification with short-A amplicons in frozen tissues. FFPE-short A: quantification with short-A amplicons in FFPE tissues. Error bars indicate the SD of the Ct values.

Mentions: To determine the quantification consistency with short amplicons, the expression profiles of the 14 target RNAs with short-A amplicons were examined in frozen tissues and compared with those of the paired long amplicons. Figure 2A intuitively shows the mean Ct values of the studied RNAs. The results demonstrated that the mean Ct values of short amplicons were significantly lower than that of long amplicons (P < 0.0001). Generally, short amplicons had 1.8 cycles less than that of long amplicons in quantitative RT-PCR, suggesting that short amplicons were more sensitive for the quantification in frozen tissues. Moreover, the short-A amplicons were evaluated in FFPE tissues from the exact CRC cases of aforementioned frozen tissues. The results presented in Fig. 2B indicated that the Ct values of short amplicons did not show significantly difference between frozen and FFPE tissues (P = 0.1455), suggesting that short amplicons quantified effectively in FFPE tissues.


Quantitative assessment of short amplicons in FFPE-derived long-chain RNA
Comparisons of Ct values obtained from short and long amplicons in frozen and FFPE tissues.(a): Comparison of Ct values obtained from short and long amplicons in frozen tissues. (b): Comparison of Ct values obtained from short amplicons in frozen and FFPE tissues. Frozen-short A: quantification with short-A amplicons in frozen tissues. FFPE-short A: quantification with short-A amplicons in FFPE tissues. Error bars indicate the SD of the Ct values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384205&req=5

f2: Comparisons of Ct values obtained from short and long amplicons in frozen and FFPE tissues.(a): Comparison of Ct values obtained from short and long amplicons in frozen tissues. (b): Comparison of Ct values obtained from short amplicons in frozen and FFPE tissues. Frozen-short A: quantification with short-A amplicons in frozen tissues. FFPE-short A: quantification with short-A amplicons in FFPE tissues. Error bars indicate the SD of the Ct values.
Mentions: To determine the quantification consistency with short amplicons, the expression profiles of the 14 target RNAs with short-A amplicons were examined in frozen tissues and compared with those of the paired long amplicons. Figure 2A intuitively shows the mean Ct values of the studied RNAs. The results demonstrated that the mean Ct values of short amplicons were significantly lower than that of long amplicons (P < 0.0001). Generally, short amplicons had 1.8 cycles less than that of long amplicons in quantitative RT-PCR, suggesting that short amplicons were more sensitive for the quantification in frozen tissues. Moreover, the short-A amplicons were evaluated in FFPE tissues from the exact CRC cases of aforementioned frozen tissues. The results presented in Fig. 2B indicated that the Ct values of short amplicons did not show significantly difference between frozen and FFPE tissues (P = 0.1455), suggesting that short amplicons quantified effectively in FFPE tissues.

View Article: PubMed Central - PubMed

ABSTRACT

Formalin-fixed paraffin-embedded (FFPE) tissues are important resources for molecular medical research. However, long-chain RNA analysis is restricted in FFPE tissues due to high levels of degradation. To explore the possibility of long RNA quantification in FFPE tissues, we selected 14 target RNAs (8 mRNAs and 6 long noncoding RNAs) from literatures, and designed short (~60&#8197;bp) and long (~200&#8197;bp) amplicons for each of them. Colorectal carcinomas with adjacent normal tissues were subjected to quantitative reverse-transcription PCR (quantitative RT-PCR) in 3 cohorts, including 18 snap-frozen and 83 FFPE tissues. We found that short amplicons were amplified more efficiently than long amplicons both in snap-frozen (P = 0.0006) and FFPE (P = 0.0152) tissues. Nonetheless, comparison of colorectal carcinomas with their adjacent normal tissues demonstrated that the consistency of fold-change trends in a single short amplicon between snap-frozen and FFPE tissues was only 36%. Therefore, we innovatively performed quantitative RT-PCR with 3 non-overlapping short amplicons for 14 target RNAs in FFPE tissues. All target RNAs showed a concordance of 100% of fold-change trends in at least two short amplicons, which offers sufficient information for accurate quantification of target RNAs. Our findings demonstrated the possibility of long-chain RNA analysis with 3 non-overlapping short amplicons in standardized-preserved FFPE tissues.

No MeSH data available.